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MIIM30002 - Principles of Immunology > Lecture 12 - Lymphocyte Activation > Flashcards

Lecture 12 - Lymphocyte Activation Flashcards

1
Q

Describe the structure of the TCR complex

A

TCR (α & β chains)

CD3: ε-, δ-, γ-chains ITAMs

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2
Q

What is important about the structure of an ITAM?

Where are they found?

A 
Tryosine residues (which can be phosphorylated) 
With several other amino acids

They are found in both:
• TCR
• BCR

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3
Q

What happens to the tyrosines in the ITAMs?

A

Phosphorylated by Src family tyrosine kinases:
• Lck
• Fyn

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4
Q

What regulates the Src family kinases?

Describe this

A

Inhibition:
1. Csk (C-terminal src kinase)
• A kinase
• Phosphorylates the tyrosine residues on Src family kinase
→ blocks the action of Src family kinase

Activation:
2. CD45
 • a phosphatase
 • removes the phosphates from the Y residues that the Csk put on
 → activates Src family kinase function
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5
Q

What is the functional relationship between Csk and CD45?

A

Direct antagonists of each other

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6
Q

What is the structure of a Src family kinase?

Outline the function of each of the domains

A

SH3 domain
• interacts with the Guanine nucleotide exchange factor

SH2 domain
• docks to other phospho-tyrosines on the receptor

Kinase domain
• tyrosine residues

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7
Q

Describe the conformation of Src-family kinases when activated and inactivated

A

Activated:
• phosphorylation of activating Tyr on kinase domain
• SH2 domain not bound by kinase domain

Deactivated:
• phosphorylation of inhibitory Tyr on kinase domain
• Kinase domain binds to SH2 domain, inactivating it

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8
Q

Describe the proximal signalling events that occur after MHC-TCR engagement

A
  1. MHC binds to TCR
  2. Recruitment of Lck with the co-receptors (CD4/CD8) to the TCR/CD3 complex
  3. Lck phosphorylates the Y-residues of ITAMs on CD3
    (these Y residues are now a docking site for ZAP-70)
  4. Recruitment of ZAP-70 to phosphorylated tyrosines of the ITAMs
  5. Phosphoyrlation of ZAP-70 by Lck
  6. ZAP-70 goes on to phosphorylate LAT and then PLC-γ
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9
Q

What is ZAP-70?

Describe its function

A
  • Syk family kinase
  • Activated by Lck (i.e. phosphorylated)
  • Phosphorylates LAT & PLC-γ
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10
Q

What is LAT?

Describe the important structural features

A

Linker of T cell activation
• Membrane bound protein

  • Contains tyrosine residues (can be phosphorylated by ZAP-70)
  • Phosphorylated tyrosines serve as docking sites for adaptors w/ SH2 domains
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11
Q

Describe the function of LAT

A

Proximal signalling events
1. Phosphorylation of LAT by ZAP-70

→ activating two different pathways:

a. MAP Kinase pathway
b. PLC-γ pathway

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12
Q

What is PLC-γ?

Describe its basic function

A

Phospholipase C-γ
• A cytosolic enzyme
• Hydrolyses membrane phospholipids

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13
Q

Describe the two pathways of PLC-γ activity

A
  1. Activated PLC-γ
  2. Production of PIP2

3a. Production of IP3
4a. IP3 increases intracellular Ca2+
5a. Ca2+ activates calcineurin (by binding to Calmodulin)
6a. Calcineurin activates NFAT through phosphatase action

3b. Induction of DAG
4b. DAG and Ca2+ activate PKC
5b. Increase in NF-κB in the cytosol

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14
Q

What does NF-κB and NFAT stand for?

A

Nuclear factor kappa B

NFAT: nuclear factor of activated T cells

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15
Q

Describe the regulation of NF-κB

A
  1. IκB bound to NF-κB, keeping it in the cytoplasm and inactivated
  2. Phosphorylation of IκB by IκB kinase, in conjunction with PKC
  3. Proteolysis of IκB in proteasome
  4. NF-κB moves into the nucleus and binds promoters
  5. Transcription of interleukins
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16
Q

What is PKC?

A

Protein kinase C

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17
Q

What is the general function of calcineurin?

A

Phosphatase - removes phosphate from NFAT

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18
Q

Why are NFAT and NF-κB regulated by location?

A

They are stuck in the cytoplasm (sequestered from the nucleus) due to:
• NFAT: phosphate
• NF-κB: IκB

19
Q

Which is the function of Cyclosporine?

A

Immunosuppressant
• Binds to calcineurin, blocking phosphatase activation

• NFAT is no longer activated by calcineurin

20
Q

Describe the MAP kinase pathway

A
  1. Phosphorylated LAT
  2. Grb-2 docks to phosphotyrosines on LAT via SH-2 domain
  3. SOS (Guanine nucleotide exchange factor) docks to SH3 of Grb-2
  4. Guanine nucleotide exchange factor catalyses
    Ras-GDP → Ras-GTP
  5. Ras activates Raf
  6. Mek
  7. Erk
  8. Elk (a transcription factor)
  9. Elk translocated to the nucleus, and activates another transcription factor (AP-1)
  10. Gene transcription
21
Q

What are some Guanine nucleotide exchange factors in the MAPK pathway?
How is it activated?
What is its function?

A

Sos
• Binds to SH3 domain of the Src family kinase (e.g. Grb-2), which was activated by the receptor

Function
• Replaces GDP with GTP in Ras, activating it

22
Q

What are the functional responses after T cells activation?

A
  • Cytokine release
  • Proliferation
  • Differentiation (Memory, effectors)
23
Q

What drives cytokine release following T cell activation?

A
  1. TCR-MHC engagement
  2. Biochemical pathway
  3. Transcription factor activation
  4. Cytokine expression
24
Q

What are the main transcription factors required for cytokine expression?
How do they function?

A
  • NF-B
  • NFAT
  • AP-1

The TFs bind to promoters of the genes for the cytokines, ‘turning on’ the gene

25
Q

Describe the variability of the TCR/CD3 complex

A

TCR: variable; cell-specific recognition domains
CD3: invariant signalling domains

26
Q

What does ITAM stand for?

A

Intracellular tyrosine-based activation motif

27
Q

What are some Src family kinases?

A
  • Lck

* Fyn

28
Q

What are the co-receptors that are involved in proximal signalling?

A
  • CD4

* CD8

29
Q

Where is Lck located?

A

Associated with the co-receptors in the cell membrane

NB Co-receptors are CD4/CD8

30
Q

What are Syk family kinases?
Give an important example of one.

Describe their structure

A

Another family of kinases (distinct form Src)

• e.g. ZAP-70

Structure:
• Tandem SH2 domains (2 x SH2)
• kinase domain

31
Q

How is ZAP-70 activated?

A

It is inactive until is interacts with phospho-tyrosines on other proteins (such as CD3 ITAMs)

32
Q

Describe the events after proximal signalling

A

At the end of proximal signalling, we have phosphorylated (an thus active) ZAP-70.

  1. ZAP-70 phosphorylates LAT
33
Q

Describe the interaction between LAT and PLC-γ

A
  1. LAT has been phosphorylated by ZAP-70
  2. PLC-γ docks on the phosphotyrosines on LAT
  3. ZAP-70 and Itk phosphorylate PLC-γ
34
Q

What is PIP2?

What is IP3?

A
  • Phosphatidyl inositol bisphosphate

* Inositol triphosphate

35
Q

What are the small G proteins in the MAPK pathway?

A

Ras (or Rac)

36
Q

What is MAP kinase kinase kinase?

A

Raf

37
Q

What is MAP kinase kinase?

A

Mek

38
Q

What is MAP Kinase?

A

Erk

39
Q

What type of molecule is Elk?

A

A transcription factor

40
Q

How does Raf activate Mek, and Mek activate Erk?

A

Phosphorylation

41
Q

In lymphocyte signalling, what is the result of the following pathways:
• MAPK
• PLC-γ & calcineurin
• PLC-γ & PKC

A

MAPK: AP-1

PLC-γ & calcineurin: NFAT

PLC-γ & PKC: NF-κB

42
Q

Which molecule activates Lck?

A

CD45

By removing the phosphate from the inhibitory tyrosine

43
Q

Which molecule inhibits Lck?

A

Csk

By phosphorylating the inhibitory tyrosine

44
Q

Which cytokine is produced through NFκB signalling?

A

IL-2

MIIM30002 - Principles of Immunology (30 decks)

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